The oil and gas industry generates large amounts of oil-derived effluents such as Heavy Crude Oil (HCO) in water (W) emulsions, which pose a significant remediation and recovery challenge due to their high stability and the presence of environmentally concerning compounds. Nanomaterials emerge as a suitable alternative for the recovery of such effluents, as they can separate them under mild conditions. Additionally, different biomolecules with bioremediation and interfacial capabilities have been explored to functionalize such nanomaterials to improve their performance even further. Here, we put forward the notion of combining these technologies for the simultaneous separation and treatment of O/W effluent emulsions by a novel co-immobilization approach where both OmpA (a biosurfactant) and Laccase (a remediation enzyme) were effectively immobilized on polyether amine (PEA)-modified magnetite nanoparticles (MNPs). The obtained bionanocompounds  were successfully characterized via DLS, XRD, TEM, TGA, and FTIR. The demulsification of O/W emulsions was achieved by MNP-PEA-OmpA and MNP-PEA-OmpA-Laccase at 5000 ppm. This effect was further improved by applying an external magnetic field to approach HCO removal efficiencies of 81% and 88%, respectively. The degradation efficiencies with these two bionanocompounds reached levels of between 5% and 50% for the present compounds. Taken together, our results indicate that the developed nanoplatform holds significant promise for the efficient treatment of emulsified effluents from the oil and gas industry. Research and development involving nanoparticles (NPs) has gained considerable attention during the last decade due to the wide spectrum of possible applications. NPs have unique properties, including nanometric size (which provides them with large surface areas), chemical stability in different environments, ease of synthesis, and low production costs . NPs hav®  -N’-ethyl carbodiimide hydro- chloride (EDC)  were used for MNP synthesis and functionalization. Tryptone, yeast extract, sodium chloride (NaCl) 98%, lactose, and sodic phosphate  agar plates containing yeast extract (5 g/L), tryptone (10 g/L), NaCl (10 g/L), and chloramphenicol (50 μg/mL). Next, a colony was inoculated in LB medium  and incubated for 16 h at 37 °C and 250 RPM. Subsequently, fresh LB medium (5 L) in a Bioflo/Celligen 115 Bioreactor  was inoculated with the previous culture and maintained for about 2 h at 37 °C, 250 RPM, and a 2 L/min air flow. The culture was grown until reaching an optical density of 0.8 at 600 nm (OD600 nm). At this point, the medium was fed with isopropylthio-β-galactoside (IPTG) (2 mM) to induce expression. IPTG exposure was performed for 5 h. Bacteria were then collected and concentrated with the aid of a 0.22 μm cassette membrane in a parallel plate filter . The collected cells with overexpressed OmpA were lysed with an ultra-sonic tip Vibra-cell VCX-750  after adding a lysis buffer . Sonication proceeded for 40 cycles  at 37% amplitude in ice. The obtained lysate medium was centrifuged at 4500 RPM and 4 °C for 2 h and the supernatant was recovered. Purification was conducted by passing the supernatant through a purification column packed with Profinity™ IMAC Resins, following the manufacturer’s instructions. Purified fractions were collected, and the presence of OmpA in each one of them was verified with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Fractions showing a single band at 35 kDa were lyophilized to preserve protein structure and stored at 4 °C until further use. Outer membrane protein A (OmpA) was recombinantly expressed in −1 with a spectral resolution of 2 cm−1 at room temperature.OmpA protein was lyophilized for 48 h at −80 °C. The final concentration of the protein was quantified by colorimetric protein determination techniques using a commercial BCA Protein Assay Kit  . The sec4 following the protocol of Feng et al. [2 precipitate resulting as byproduct of the oxidation reaction was filter under vacuum with a 0.7 µm paper filter until obtaining a transparent solution. The pH of the sample was adjusted to 2.0 by adding 10 M of hydrochloric acid (HCl) dropwise. Excess KMnO4 and HCl was removed by dialysis using a 100–500 Da cut-off molecular weight membrane with ultra-pure water for 12 h. Finally, the water excess in the PEA solution was removed in a rotary evaporator  under vacuum at 80 °C for 6 h. The PEA oxidation was verified by FTIR, and its approximate concentration was determined by the chemical titration of the carboxyl groups . A total of 3 g of Jeffamine M-600 polyetherimine (PEA) was oxidized in 50 mL of a 0.015 M solution of KMnOg et al. . The mix2.4H2O (2.73 mmol) and 7.56 g of FeCl3.6H2O (27.96 mmol) were dissolved separately in 40 mL of ultra-pure water to obtain 0.3 M and 0.7 M solutions, respectively. Additionally, 5.6 g of NaOH were dissolved in 40 mL of water. Chlorides solutions were then mixed in the presence of 40 mL of ultra-pure water and 2 mL of 2% (v/v) solution of TMAH (to avoid agglomeration), followed by heating up to 90 °C. Finally, the NaOH solution was manually dropped into the chloride mixture at a rate of 3 mL/min while maintaining vigorous agitation at 1500 RPM and the temperature at 90 °C. The reaction was carried out for 1 h. The obtained MNPs were washed at least 3 times with ultra-pure water with the aid of a strong permanent magnet for rapid separation and sonicated in an ultrasonic bath  in between washes.Magnetite nanoparticles were synthesized by the co-precipitation method. Briefly, 2.528 g of FeCl−1 with a spectral resolution of 2 cm−1.The size of the obtained MNP was measured using a Dynamic Light Scattering (DLS) instrument  and Transmission Electron Microscopy (TEM) . Additionally, the purity of the crystalline phase of magnetite was verified via X-Ray Diffraction (XRD). Finally, effective synthesis was confirmed by Fourier-transform infrared spectroscopy (FT-IR) and thermogravimetric analysis (TGA) . Infrared spectra were recorded using an A250/D FT-IR . The FT-IR parameters were specified with the aid of the OPUS Spectroscopy Software . Spectra were recorded in the range of 4000–400 cmv/v) solution of TMAH was added to help resuspending the MNP. Then, 20 µL of 99% glacial acetic acid was added prior to the conjugation of APTES (200 µL). This compound rendered free amine groups on the surface of the MNPs. The reaction was carried out at 60 °C and 250 RPM for 1 h. Silanized nanoparticles were washed five times with ultra-pure water and 1.5% (w/v) NaCl solution to remove excess APTES, aided by a strong permanent magnet.The silanization of MNP was carried out with an initial sample of 100 mg of MNP in 30 mL of ultra-pure water. The process was started by sonicating the MNP for 40 min to avoid agglomeration. Next, 200 mL of a 2.5% (v/v) of glutaraldehyde (GA) was added to the MNP sample and left under mechanical agitation at 250 RPM and room temperature for 24 h. Oxidized PEA was then conjugated to the obtained MNP-AP-GA by adding 1 mL of PEA solution in a 1:2 molar ratio with respect to the free amine groups. The reaction proceeded overnight at 250 RPM and room temperature. Finally, the PEA-coated magnetite nanoparticles were washed three times with ultra-pure water and three times with 1.5% (w/v) NaCl solution.Silanized MNP was resuspended in 30 mL of ultra-pure water and sonicated for 40 min. Then, 2 mL of 2%  by a Bruker Alpha II FTIR Eco-ATR . Spectra were collected in the range of 4000–400 cmlization . TGA wasThe O/W emulsion formulation was based on previous studies by Aburto et al. ,19. The Stable emulsions were prepared as follows: (i) A volume of brine solution, corresponding to 97% of the total volume of the sample, was transferred to a vessel and heated to 60 °C in a hot plate. This temperature was kept constant throughout the entire experiment. (ii) In a separate hot plate, 0.6 mL of heavy crude oil was transferred to a vessel and heated to 60 °C. A homogenizer was then connected to the hot brine container, which was equipped with a propeller located such that a ratio of propeller (Da) to vessel diameter (Dt) of about 1  was maintained see . (iii) A® (https://imagej.nih.gov/ij/). Bottle testing is a widely used protocol for the easy assessment of demulsifier performance in oilfields. The incorporation of demulsifier at different concentrations is required, considering the significant amount of water added to homogenize it. The mixture was then added to the dispersed phase. Traditional bottle tests were carried out to determine the stability of the O/W emulsion. The stability of HCO in water (O/W) emulsions was measured by following the changes in transmission signals and backscattering of light over time. This was also useful to assure the reproducibility of the emulsion manufacturing protocol. The Δ backscattering (ΔBS) measurement and the Turbiscan Stability Index (TSI) were recorded in a Turbiscan Lab Stability Analyzer  at conditions of transmission (0° from the incident light beam) and backscattering (135° from the incident light beam) by utilizing a pulsed near-infrared light source (λ = 850 nm). For this purpose, samples were carefully transferred to a glass vial by preventing bubble formation at the top of the container. Measurements took place every minute for 1 h at room temperature (25 °C). HCO drops were imaged in an optical microscope with a 100X objective (NA = 40). Image analysis was performed in the open access software Image JThe destabilizing effect of MNP and OmpA on the formulated O/W emulsion was evaluated separately in the Turbiscan for 1 h at room temperature. For this purpose, 16 mL of the O/W emulsion were placed in a glass vial along with 200, 500, 1000, or 5000 ppm  of MNP or OmpA. The mixture was then homogenized at 100 RPM. The ΔBS was monitored to determine the range of bionanocompound concentrations required for effective HCO removal. TM 10S, Thermo Fisher Scientific TM, Waltham, MA, USA) at 225 nm, where the HCO exhibits a characteristic peak [An effective concentration of 5000 ppm (5000 µg/mL) of OmpA and MNP for HCO removal was determined. Concentrations of bionanocompounds above 5000 ppm failed to produce a further improvement in separation (data not shown). Such optimal concentration corresponded to an effective treatment ratio of 1:5 . Consequently, the demulsification tests with MNP-PEA-OmpA and MNP-PEA-OmpA-Laccase bionanocompounds were conducted at this concentration. After the incorporation of the bionanocompound, the sample was left at rest for 1 h to monitor separation by direct observation. Next, the bionanocompounds were recovered by placing a neodymium permanent magnet (1.2 T) at the bottom of the glass vial. Even though the bionanocompounds were easily recovered after treatment, further studies will be required to assess whether they could be recycled into the process. Subsequently, the remaining HCO in the sample was extracted with petroleum ether at a 1:3 mass ratio (sample:ether) in an ultrasound bath for 5 min. The non-polar phase with the HCO dissolved in ether was collected and analyzed via UV-VIS spectrophotometer  in an Agilent technologies  6890N gas chromatography instrument coupled to a selective mass detector (MSD) 5975 in Full Scan mode. The GC was equipped with a HP-5MS column (30 m × 0.325 m × 0.25 μm) operating at a 1.2 mL/min flow rate. The initial oven temperature was held at 35 °C for 5 min. It was then increased at a rate of 8 °C/min to 220 °C and kept there for 5 min for a total detection time of 33.125 min. The injection mode was pulsed splitless at a 10 mL/min flow rate of Helium as entrainment gas at 200 °C. The role of the Laccase was obtained by comparing the GC-MS results obtained with the full bionanocompound  with those obtained with the MNP-PEA-OmpA bionanocompound. Prior to injection into the GC-MS system, the sample was extracted with dichloromethane and concentrated to 1 mL with N−1 and 1513 cm−1 are associated with the Amide I and Amide II bands, respectively. The peak at 1260 cm−1 corresponds to the C-O stretching vibrations. The peaks at 1059 and 3200 cm−1 can be attributed to the N-H stretching bands vibration [The purity of the obtained protein was approximately 94%. ibration . The secibration . Lanes 23O4 were identified  and corroborated the absence of precursor impurities and unwanted crystalline phases such as Hematite [Hematite . TEM imaHematite .−1, which can be attributed to the C-O-H stretching vibration and an additional band at 1471 cm−1, which can be correlated with the C-H bending vibration. Compared to the PEA IR spectrum, oxidized PEA exhibits three different vibrational bands at about 1645, 1370, and 1304 cm−1, which can be explained by the presence of C=O stretching, C-H bending, and O-H stretching, respectively. Of interest are the C=O stretching bands at 1645 and 3350 cm−1, because they can be associated with the COOH bonds of carboxyl groups needed for the conjugation of OmpA and Laccase. The peak at 2867 cm−1 corresponds to the amorphous PEA (C-H asymmetric stretching) [PEA oxidation was confirmed by Fourier transformed infrared spectroscopy (FTIR). etching) .−1, which can be attributed to the Fe–O bonds of iron oxide. This band continues to be present along all conjugation steps [−1, the bending vibrations of C–N and C–H at 1325 cm−1 and 1513 cm−1 as well as N–H bands at 1558 cm−1 and 3741 cm−1 confirm the silanization with APTES (MNP-AP). The conjugation of PEA on silanized magnetite (MNP-AP-PEA) was confirmed by the absorption peak at 1041 cm−1 for the C–H bending vibrations and at 1690 cm−1 related to the presence of C=O stretching in the backbone of the polymer. Finally, the conjugation of OmpA and Laccase on the surface of MNP (MNP-PEA-OmpA-Laccase) was verified by the presence of the amide I and II bands at 1651 and 1513 cm−1, respectively.The hydrodynamic diameter of the bionanocompound is reported in on steps . The pre−1. Additionally, a-helix structure and 310-helix were identified at 1655 and 1664 cm−1, respectively [The secondary structural changes of immobilized OmpA were assessed with the aid of the second derivative of the FTIR spectra . The resectively . After iThe conjugation efficiencies after each functionalization step were assessed with the aid of thermogravimetric analysis (TGA). TGA thermograms for bare magnetite (MNP) and the MNP-PEA-OmpA, MNP-PEA-Laccase, and MNP-PEA-OmpA-Laccase bionanocompounds are shown in As a well-known oxide-reductase, the enzymatic activity of Laccase can be easily determined by measuring the rate of oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) . For thiMNP or 0.22 mg/mgMNP  was 0.12 mg/mgMNP, while for the MNP-PEA-OmpA-Laccase (97.44% yield) it was 0.06 mg/mgMNP.The enzyme contents were estimated based on the enzymatic activity as described in mg/mgMNP . SimilarOne of the most important properties of MNPs is the interfacial activity, which can be further improved by functionalization with interfacially active molecules—e.g., amphipathic surfactants. Several other materials have been coated on the surface of MNP to improve their porosity, stability, or affinity for specific interfaces . ExampleThe physical stability of the prepared O/W emulsions was followed by monitoring the change in transmission signal and backscattering at room temperature in a Turbiscan Analyzer. This approach allowed quantification of destabilization phenomena such as sedimentation, creaming, aggregation, flocculation, and coalescence. The Turbiscan Stability Index (TSI) was also determined as a measure of the relative importance of the destabilization phenomena . Low valOmpA and MNP were added to the emulsion at concentrations of 200, 500, 1000, and 5000 ppm and their destabilizing effect was recorded with the aid of a Turbiscan instrument. We explored increased concentrations of demulsifier to analyze whether destabilizing phenomena might occur at a window of higher concentrations. The ΔBS results for O/W emulsions in the presence of OmpA and MNP are shown in This agrees well with previous studies where superior stabilization is achieved by mixing nanoparticles with chemical surfactants in systems known as Pickering emulsions ,83. ThisWe also tested MNP-PEA-OmpA and MNP-PEA-OmpA-Laccase for demulsification and estimated their remediation capabilities by looking at the changes in chemical composition via GC-MS. Under this perspective, we conducted bottle tests with MNP-PEA-OmpA and MNP-PEA-OmpA-Laccase bionanocompounds at a concentration of 5000 ppm. This corresponds to an approximate concentration of 600 ppm of OmpA for the MNP-PEA-OmpA bionanocompound. In the case of the MNP-PEA-OmpA-Laccase bionanocompound, the corresponding concentrations were 300 ppm for OmpA and 550 ppm for Laccase. Finally, by assuming that all Laccase molecules remain active after immobilization, we calculated a catalytic activity of 18.4 U. ® software. Each sample was analyzed in triplicate at different z depths to find an average particle size distribution that approached 100–250 nm (not shown). Such an average size has been typically observed for miniemulsions or nano-emulsions, which generally exhibit superior stability and tolerance against sedimentation or creaming processes [A representative optical microscopy image of crude oil-in-water (O/W) emulsions prepared in this work is shown in rocesses .Upon the recovery of the bionanocompounds from the treated samples, we measured the HCO remaining in them. Briefly, 1 mL of residual water was mixed with 3 mL of petroleum ether and sonicated in an ultrasonic bath for 5 min. The mixture was decanted to separate the polar (water) from a non-polar phase (ether). The HCO preferentially partitioned into the ether phase, which was collected and quantified spectrophotometrically in a UV-VIS instrument at 225 nm (HCO detection wavelength ). ExtracMNPs have the potential to enable numerous industry applications as their exceptional surface area and amenability for functionalization facilitate coupling of molecules with an ample range of functionalities. One of such applications is the separation of oil-water emulsions where the MNPs increase the density of the water present in the emulsion, thereby accelerating the rate of water settling. Additionally, cationic surfactant-coated MNPs have demonstrated that oil droplets are successfully separated from the negatively charged oil-in-water emulsions. This is in contrast with anionic surfactant-coated MNPs that showed no interfacial activity, thereby indicating that the stable adsorption of MNPs at the water/oil interface caused by electrostatic attractions plays a vital part in effective separation ,86. AddiThe treated O/W emulsions were also analyzed by GC–MS analysis to semi-quantitatively estimate changes in the composition of HCO with respect to the blank—i.e., untreated—O/W emulsion. Chromatograms of crude oil are shown in The relative areas of peaks for compounds in the treated samples were estimated with respect to the highest intensity peak in the blank . Particulate materials have played a fundamental role in the oil and gas industry due to their ability to stabilize or destabilize colloidal systems. As a result, since the beginning of the century they have found several applications ranging from the formulation of drilling fluids to the treatment of effluents . With thWe synthesized the MNPs by chemical co-precipitation and confirmed a diameter of about 80 nm by DLS and TEM. Functionalization and protein immobilization were successfully confirmed via FTIR and TGA. The estimated efficiency of total protein immobilization approached about 2.5%, 5.0%, and 5.5% for the MNP-PEA-OmpA, MNP-PEA-Laccase, and MNP-PEA-OmpA-Laccase bionanocompounds. These results suggested that immobilization took place under possible competing protein–protein and protein–surface interactions. The immobilization yield for the Laccase alone after co-immobilization (MNP-PEA-OmpA-Laccase) was estimated by a colorimetric assay based on ABTS to be >97%.Demulsification assays were conducted for O/W 1 wt% emulsions. Separation was achieved at 5000 ppm of the MNP-PEA-OmpA and MNP-PEA-OmpA-Laccase bionanocompounds. This was further improved by applying an external magnetic field to reach HCO removal efficiencies of 81% and 88%, respectively. These experiments demonstrated that the bionanocompounds can be recovered after treatment, which opens the possibility for potentially recycling them back into the process. The demulsifier activity of the recovered bionanocompounds needs to be assessed in detail.The potential of the bionanocompounds in the bioremediation of emulsified effluents was tested by measuring changes in the composition of the selected compounds. The results indicate that, in the presence of the MNP-PEA-OmpA and MNP-PEA-OmpA-Laccase bionanocompounds, we observed degradation efficiencies in the range of 5% to 50%. The highest degradation efficiencies were for heptadecane and hexadecane 2,6,10,14-tetramethyl, which approached 25% and 50%, respectively. For future studies, we recommend the use of internal standards for a more quantitative determination. Overall, our results hold significant promise for the simultaneous separation and treatment of emulsified effluents from the oil and gas industry. Moreover, due to the remaining magnetic susceptibility, chances are high that they can be incorporated into a separation/treatment unit operating cyclically.


Heart rate variability (HRV) is a valid and non-invasive indicator of cardiac autonomic nervous system functioning. Short-term HRV recordings  produce data that usually is manually processed. Researcher subjective decision-making on data processing could produce inter- or intra-researcher differences whose magnitude has not been previously quantified in three independent human cohorts. This study examines the inter- and intra-researcher reproducibility of HRV parameters  derived from short-term recordings in a cohort of children with overweight/obesity, young adults and middle-age adults. Participants were recruited from 3 different studies: 107 children , 132 young adults  and 73 middle-aged adults . HRV was measured using a Polar RS800CX heart rate monitor. The intraclass correlation coefficient (ICC) ranged from 0.703 to 0.989 and from 0.950 to 0.998 for inter-and intra-researcher reproducibility, respectively. Limits of agreement for HRV parameters were higher for the inter-researcher processing compared with the intra-researcher processing. On average, the intra-researcher differences were 31%, 62%, and 80% smaller than the inter-researchers differences based on Coefficient of Variation in children, young and middle-aged adults, respectively. Our study provides the quantification of the inter-researcher and intra-researcher differences in three independent human cohorts, which could elicit some clinical relevant differences for HRV parameters. Based on our findings, we recommend the HRV data signal processing to be performed always by the same trained researcher and we postulate a development of algorithms for an automatic ECG selection. Both autonomic branches  are considered the main determinants of the magnitude of HRV3. However, from an integrative physiological point of view, the HRV has been considered an indicator of the complex inter-relationship between different physiological systems 4. Specifically, HRV refers to the variability of time intervals between normal consecutive heart beats1. Usually, a healthy heart shows higher beat-to-beat variability  compared with a less healthy heart6. In consequence, a reduced HRV is related to a higher morbidity , and mortality8. Furthermore, a decrease in HRV has been related to ageing processes, especially from 40 to 60 years9.Heart rate variability (HRV) is a valid and non-invasive indicator of cardiac autonomic nervous system functioning10. These instruments have been usually employed to collect data during long-term HRV recordings (24-h), short-term HRV recordings (5–15 min) and ultra-short-term HRV recordings (< 5 min)11. Short-term HRV recordings at rest have gained popularity in recent years due to their high feasibility together with the capacity to obtain meaningful and accurate data. Moreover, short-term HRV recordings make easier the HRV signal acquisition under more controlled conditions  compared with long-term HRV recordings11.HRV can be accurately recorded using non-invasive tools such as an electrocardiogram or a heart rate monitor12. Data processing of HRV recordings allows to partially remove both mechanical and physiological artefacts from the HRV signal recorded 12. Usually, the HRV recordings are screened to manually select the 5 top-quality minutes14, which is defined as: (1) no presence of large R–R interval outliers, (2) equidistance between consecutive R–R intervals and (3) unimodal Gaussian R–R intervals and heart rate distribution graphics14. Thus, the HRV data signal processing of short-term recordings requires, in a greater or lesser extent, subjective decisions from the researcher, which could lead to inter- and intra-researcher differences of HRV parameters derived from short-term recordings.Despite recording advantages previously commented, the HRV data signal processing of short-term recordings can affect the HRV derived parameters afterwards16. Farah et al.15 found that the inter-researcher reproducibility was lower than the intra-researcher reproducibility in 27 healthy adolescents and concluded that the same researcher should perform the HRV data signal processing of short-term recordings15. In the same way, Bassi et al. reported similar results to the obtained by Farah et al.15 in a cohort of 44 middle-aged adults with type 2 diabetes16. However, little is known about how much the observers’ decisions influence HRV parameters measured with heart rate monitors  in different age-range populations including a relatively larger sample size than in the previous studies16. This is even more important since such heart rate monitors are getting more and more popular not only among researchers but also among common users. Importantly, several aspects may affect the reproducibility of HRV measurements, such as individual characteristics17 or technical aspects18. Therefore, the present work examines whether the inter- and intra-researcher reproducibility of HRV parameters derived from short-term recordings using the Kubios software has an impact on the HRV derived parameters  in three different cohorts .To our knowledge, only two previous studies have tested either the inter- or intra-researcher reproducibility of the selected R–R interval period using the Kubios software19, 132 young adults  from the ACTIBATE study20 and 73 middle-aged adults  from the FIT-AGEING study21. The inclusion/exclusion criteria have been described elsewhere21. Briefly, the inclusion criteria in common for the three studies were: (1) not being physically active; (2) not having a diagnostic of cardiovascular disease; (3) not being a smoker; (4) not being enrolled in a weight loss program; and (5) not being pregnant.This study used baseline data from three different studies. Participants were 107 children with OW/OB  from the ActiveBrains projectThe ActiveBrains, ACTIBATE and FIT-AGEING studies were approved by the Committee for Research Involving Human Subjects at the University of Granada , Servicio Andaluz de Salud  and the Human Research Ethics Committee of the “Junta de Andalucia” [0838-N-2017] for the FIT-AGEING study. These projects were registered in the ClinicalTrials.gov , and conducted according to the Declaration of Helsinki (revision of 2013). All participants and parents (if subjects were under 18) were informed of the purpose of the study and written informed consent was obtained.2. Body weight, height and waist circumference were measured twice consecutively by the same trained researcher, and the average for each parameter was used.Body weight and height were measured with an electronic scale and a stadiometer, respectively  prior to the HRV assessment. Body mass index (BMI) was calculated as kg/mStudy design is graphically presented in Fig. 18 using the Kubios software, standard version 3.2 23. The R–R intervals series were detrended using the smoothness prior method with lambda set at 500 and a cubic interpolation at the default rate of 4 Hz. A visual inspection was performed on the 10–15 min recorded, with each researcher manually selecting 5 min of the register  after considering that the selected 5 min met the top-quality criteria: (1) no large R–R interval outliers , (2) R–R intervals equidistance , and (3) Gaussians R–R intervals and heart rate distribution graphics14.Participants lay in a supine position for 10–15 min while the HRV signal was recorded. HRV assessment took place in a quiet room with dim lighting, controlled ambient temperature and humidity (ACTIBATE and FIT-AGEING studies) between 8 AM and 12 PM . The Polar RS800CX heart rate monitor  was used to record the HRV signal during 10–15 min at a sampling frequency of 1,000 Hz. Participants were instructed to breathe normally, and not to fidget, talk or sleep during recordings. For HRV signal analyses we used the normal R–R intervals after excluding the extreme values with a medium filter1, we derived the most used time- and frequency-domain HRV parameters obtained from short-term recordings. In the time-domain, we computed the squared root of the mean of the sum of the squares of successive normal R–R interval differences (RMSSD) in milliseconds (ms), number of pairs of adjacent normal R–R intervals differing by more than 50 ms in the entire recording, expressed as a percentage (pNN50), and the standard deviation of all normal R–R intervals in ms (SDNN). In the frequency-domain, we performed spectral analyses using the non-parametric and parametric methods, fast Fourier transformation (FFT) and autoregressive (AR) algorithms respectively. FFT algorithm with Welch’s periodogram method  and AR with a model order of 16 as previously recommended24. We derived the power in the high frequency (HF: 0.15–0.4 Hz), the power in the low frequency (LF: 0.04–0.15), both in absolute units (ms2) and the LF/HF ratio.Under the Guidelines of Task Force of The European Society of Cardiology and The North American Society of Pacing and Electrophysiology recommendations7. However, the time-domain HRV parameters measure autonomic nervous system activity but cannot differentiate changes on HRV due to withdrawal of vagal tone or increased sympathetic tone and are less precise in terms of frequency delimitation3. On the other hand, HRV parameters in frequency-domain can quantify cyclic fluctuations of R–R intervals. The spectral analyses divide HRV into different oscillatory components (such as HF or LF). HF component reflects respiratory sinus arrhythmia, which is mainly influenced by vagal activity. LF fluctuations are due to parasympathetic and sympathetic control. The ratio LF/HF is often considered as an indicator of sympatho-vagal balance , however, this interpretation has been recently challenged25.The physiological interpretation of HRV parameters in time- and frequency-domain should be briefly mentioned. Usually, higher HRV values on time-domain parameters  are considered as higher vagal tone associated with a lower cardiovascular disease risk and mortality26 and the root mean square error (RMSE) were used to study the inter- and intra-researcher reproducibility for HRV parameters derived from short-term recordings. Heteroscedasticity, or whether the variability (error) increases or decreases as the magnitude of the measure increase, was calculated using linear regressions between the mean values and the absolute values of HRV parameters differences. Of note, all the analyses were performed separately because we observed an interaction age × study population effect, influencing most of the HRV parameters (P < 0.05). All the analyses were conducted using the Statistical Package for Social Sciences .Descriptive data are presented as means and standard deviations (SD) for continuous variables, and frequencies and percentages for categorical variables. Normal distribution of the included variables was tested using the Kolmogorov–Smirnov test, and visual inspection of histograms was also performed. HRV parameters derived from short-term recordings did not exhibit normal distributions, however, since sensibility analyses did not show differences after normalizing the variables, data were analyzed and presented as non-normalized values. Intraclass correlation coefficient (ICC) confidence interval (95% IC), inter- and intra-coefficient of variation (CV), Bland–Altman plots (mean difference [bias] and limits of agreement)Table Table Figures 27 based on limits of agreement in Bland Altman plots); (2) overall, HRV parameters in frequency-domain derived from short-term recordings presented lower reproducibility compared with those in time-domain in both the inter- and intra-researcher processing. Therefore, the data obtained from our three cohorts support the idea that HRV signal processing should be performed by the same trained researcher to obtain consistent and reproducible HRV parameters derived from short-term recordings in time- and frequency-domain. Alternatively, automatic algorithms for an ECG segment selection should be developed to overcome the reproducibility problem.The main findings of our research are: (1) this study provides the quantification of the inter-researcher and intra-researcher differences  in three different human cohorts. Although the inter- and intra-researcher differences were not very large, the inter-researcher reproducibility could elicit clinical relevant differences for some HRV parameters  and performed the same statistical tests to study inter-and intra-researcher reproducibility of HRV parameters derived from short-term recordings.To our knowledge, only three previous studies focused on inter- and intra-researcher reproducibility for HRV derived parameters from long-term recordings15 carried out in adolescents, ICCs ranged from 0.915 to 0.996 and 0.990 to 0.993 for inter- and intra-researcher reproducibility, respectively, while for children with OW/OB our ICCs ranged from 0.823 to 0.989 and 0.950 to 0.998 for inter- and intra-researcher reproducibility, respectively. Likewise, CVs reported by Farah et al.15 were lower than 20% and 7% for inter-and intra-researcher reproducibility, respectively. We found similar CVs in children, i.e., lower than 15.9% and 4.3% for inter-and intra-researcher reproducibility, respectively. The marginal differences observed across studies could be partially explained by the different biological characteristics’ between them  and/or by some methodological inconsistencies in HRV data collection  and/or HRV data signal processing.In the study conducted by Farah et al.16 reported ICC ranging from 0.730 to 0.970 and 0.910 to 0.990 for inter- and intra-researcher reproducibility in middle-aged adults with type 2 diabetes. The little variation observed of ICCs between both studies in middle-aged adults could be partially explained by the health status of the study populations  and by some methodological considerations, e.g., different model of the heart rate monitors used (Polar RS800CX vs. Polar S810i model).In regards to young adults, in our study, ICCs ranged from 0.822 to 0.985 and 0.990 to 0.996 for inter- and intra-researcher reproducibility respectively. Likewise, the CVs were lower than 16.7% and 10.9% for inter-and intra-researcher reproducibility. On the other hand, respect to middle-aged adults, ICCs ranged from 0.850 to 0.987  and 0.951 to 0.995 for inter- and intra-researcher reproducibility, respectively. Similarly to our findings in healthy middle-aged adults, Bassi et al.The inter-researcher reproducibility of HRV parameters derived from short-term recordings was lower than intra-researcher reproducibility in our study, which could be explained by one issue related to the HRV data signal processing. Based on our previous experience in the HRV data signal processing from short-term recordings using the Kubios software, we found that, in HRV recordings presenting a high HRV , a Gaussian distribution of R–R intervals and HR distribution graphs is difficult to be found. Moreover, this higher HRV could complicate the decision-taking from researchers that guarantee the selection of the same best-quality 5 min, and this fact could produce the mentioned differences between HRV parameters derived from short-term recordings.In regards to intra-researcher reproducibility, we reported CVs lower than 4.3%, 10.9% and 13.7% for children with OW/OB, healthy young and middle-aged adults respectively, in some of the HRV parameters derived from short-term recordings. Thus, in general the intra-researcher reproducibility was higher  in children with OW/OB in comparison with young and middle-aged adults. We expected that individuals with a higher HRV  presented worse intra-researcher reproducibility, in other words a higher variability between test and retest, than in individuals with lower HRV values . Hence, a higher HRV might complicates the selection of the same best-quality 5 min across researchers. In fact, in our study, the differences observed across cohorts in the intra-researcher reproducibility could be explained, in a greater or lesser extent, by methodological aspects, e.g., a different researcher performs the intra-researcher HRV signal data processing in each cohort 34. For example, for RMSSD inter-and intra-researcher HRV signal data processing, we reported a mean difference of − 1.71 ms and 0.32 ms in children with OW/OB, respectively. Thus, based on the previous studies34, we can assume that these differences are not clinically relevant. However, limits of agreement describe inter-researcher differences up to 17.31 ms, i.e., twice the change reported in the previous mentioned interventions34, reaching intra-researcher differences a maximum of 5.15 ms. These findings question whether the nature of the change reported in the intervention could be produced by the intervention itself in a same extent that it could be produced by unreliable HRV data signal processing. To define the trained level or experience of the researchers and how many researchers were in charge of the HRV data signal processing in each time point of an intervention is a matter of main interest that should be reported in all the intervention studies.Despite those methodological issues, it is relevant to take into consideration the absolute values of inter-and intra-researcher differences . Importantly, the mean values of the inter- and intra-researcher differences for SDNN in our study were 1.05–1.69 ms and 0.04–0.50 ms, respectively. However, the limits of agreement showed that some inter-researcher differences could reach 11.28 ms in young adults, where intra-researcher analysis can reach a maximum difference of 6.59 ms. Thus, attending to our findings, inter-researcher differences could be clinically relevant whereas intra-researcher differences could not.Otherwise, Bilchick et al.We encourage researchers to report the HRV signal data processing in detail  including if the same or a different researcher carried out the HRV processing, as well as their experience . Further studies are needed to test the impact of other decisions related to the HRV signal data processing such as different levels of artefact correction or experience status of the researchers on the quantification of HRV derived parameters.35.Some limitations need to be mentioned: (1) the sample from ActiveBrains project were children with OW/OB, so we cannot extrapolate our findings to thinner children; (2) the intra-researcher HRV signal data processing was not performed by the same researcher in the three different cohorts, however, all the researchers in our study were trained in HRV signal data processing using the Kubios software; (3) the experimental conditions during the data acquisition were not identical in the different studies as different standard operation procedures were used . On the other hand, the strengths of our study were: (1) relatively larger sample size compared to previous studies that analyzed inter- and intra-researcher reproducibility of HRV parameters derived from short-term and long-term recordings; (2) inter- and intra-researcher reproducibility of HRV parameters derived from short-term recordings were tested in three different cohorts and performing an identical methodology of HRV signal data processing and following the current guidelines for HRV assessmentIn conclusion, our study provides the quantification of the inter-researcher and intra-researcher differences  in three different human cohorts. Although the inter- and intra-researcher differences were not very large, the inter-researcher reproducibility could elicit some clinically relevant differences for some HRV parameters in time-domain. Based on our findings, we recommend that the HRV signal data processing should be performed by the same trained researcher to obtain more reproducible and consistent HRV parameters derived from short-term recordings. Alternatively, automatic algorithms for an ECG segment selection should be developed to overcome the reproducibility problem. These findings need to be considered with caution due to the three different cohorts selected from three studies with different objectives and inclusions criteria.Supplementary information


The aim of this study was to assess occupational exposure to diisononyl phthalate (DiNP) and di(2-propylheptyl) phthalate , the plastic producing company used both DPHP and DiNP, and the latter two companies used DiNP in their production. Exposure was assessed by measuring phthalate metabolites in urine samples (biomonitoring) and by performing air measurements. Low-level occupational exposure to DiNP was observed in the company that produced coated textiles—out of eight workers, one extruder operator was exposed to DiNP at levels exceeding the non-occupationally exposed population background levels. Some workers in the cable factory and the plastics producing company were occupationally exposed to DPHP. Air levels of phthalates were generally low, mostly below the limit of quantification. All phthalate metabolite concentrations were, however, well below the calculated biomonitoring equivalents, which suggests that the health risks related to the exposure are low. Phthalates  are ubiquitous environmental contaminants that may cause adverse effects on reproduction via their endocrine-disrupting properties. Environmental phthalate exposure has been extensively studied in various countries—even relatively long exposure time-trends are available for many phthalate metabolites in urine samples. Occupational phthalate exposure has been studied much less . This isThe data on occupational DiNP exposure consists of five studies ,3,4,5,6.p < 0.0001). Hines et al. also studied the exposure of 12 workers in PVC compounding. Their post-shift urinary results (GM 9.05 µg/L and data range 1.86 to 19.8 µg/L) were statistically different from the mid-shift results of the same workers (p = 0.03). These results indicate occupational DiNP exposure of the PVC workers studied.Two occupational studies exist that have analyzed the secondary metabolite monocarboxy isooctyl phthalate  in post-shift samples of workers. The geometric mean (GM) urinary cx-MiOP concentration of seven workers who used DiNP on polyvinyl chloride (PVC) film manufacturing was 50.5 µg/L (data range 15 to 164 µg/L) . The resKoch et al. studied five PVC plastisol workers in Germany . The uriData on occupational DiDP and DPHP exposure are scarce. Koch et al. studied DiDP/DPHP metabolites in the urine of PVC plastisol workers in a car factory in Germany . The worThe aim of this study was to assess phthalate exposure among plastics workers in Finland. Human biomonitoring was the main method for assessing exposure, but we also carried out some air measurements. We compared the urinary levels of workers to the background urinary levels of occupationally non-exposed population  and to bn = 5), a plastic producing company (n = 5), a producer of coated textiles (n = 8), and a tarpaulin producer (n = 2). The cable factory used DPHP (CAS 53306-54-0), the plastics producing company used both DPHP (CAS 53306-54-0) and DiNP , and the latter two companies both used DiNP (CAS 28553-12-0) in their production. Diisodecyl phthalate  was not used during the sample collection campaign, and other phthalates were not used because of the phthalate restriction.Potential companies for the study were contacted via telephone or email. Four companies took part in the study: A cable factory . Three men were smokers.The control population consisted of 60 occupationally non-exposed volunteers  from the Helsinki, Kuopio, and Tampere regions. One spot urine sample (first-morning void) was collected from these volunteers .The study design was given to all the participants in written form, and each gave their written consent. The study was approved by the local Coordinating ethics committee  (reference 347/13/03/00/149).The participants were given instructions to collect 5 spot urine samples as follows. (i) First-morning-void after a 2‒4-day holiday. Since phthalates have a short biological half-life ,10, we eThe sample collection took place in 2015. The urine collection vessels and storage vessels were made of polypropylene, polystyrene, polyethylene, or glass. The urine samples were stored as frozen until analysis (−20 °C).d = (1.021−1)/[d(sample)−1], where the d(sample) was the specific gravity of the urine sample. The value of 1.021 is the average urine specific gravity value of the Finnish working-age population . In statistical calculations, we replaced urinary values below LOQ with values of LOQ/2 [In order to take urinary dilution into account, the phthalate metabolite concentrations were normalized to specific gravity and adjusted to creatinine excretion. Normalization was carried out by multiplying the original concentration with correction factor kof LOQ/2 . BecauseThe native and isotope-labeled standards for MiNP were acquired from Cambridge Isotope Laboratories, Inc. . The native and isotope-labeled standards for cx-MiOP and cx-MiNP were purchased from Santa Cruz Biotechnology, Inc. , and for OH-MPHP from Toronto Research Chemicals, Inc. . The mehanol was purchased from J.T. Baker , and the acetic acid was purchased from VWR International Ltd. . The glucuronidase/sulphatase enzyme solution  was obtained from Sigma-Aldrich . The diisodecyl phthalate was purchased from Chiron AS , and the diisononyl phthalates from LGC Standard GmbH  and Chiron AS. The internal standard D4-diisononyl phthalate and di(2-propylheptyl) phthalate were from Sigma Aldrich, and the formic acid up to 98% from Fluka . The structures of phthalates and their metabolites are provided in Total phthalate metabolite concentration was measured (free + conjugated). For quantitation at the beginning of the analytical procedure, isotope-labeled internal standards for phthalate metabolites (5.0 ng of each) were added to the 0.25 ml urine sample. The sample volume was adjusted to 0.50 ml with water, consisting of 0.05 µL glucuronidase/sulphatase solution . The enzyme reaction (deconjugation) took place at 37 °C for 20 h. Next, the hydrolysis sample was centrifuged using Eppendorf 5810  at 2600 g for 10 min. Prior to the instrumental analysis, 100 µL of the hydrolyzed sample was transferred to an autosampler vial, and 10 µL of 1% aqueous acetic acid was added. Phthalate metabolites were separated and quantified using the Thermo Scientific UltiMate 3000 Rapid Separation LC system  connected to a Thermo Finnigan TSQ Quantum Discovery MAX triple quadrupole mass spectrometer . The details of the LC-MS/MS conditions are given in The air samples were collected at the workplaces to measure DiNP, DiDP, and DHPH concentrations during the production processes. Stationary and/or personal breathing zone (PBZ) samples were collected into Tenax TA® OVS tubes . OVS samplers can collect both particulate and gas-phase compounds. Calibrated pumps were used in sample collection with an airflow of 1 l/min. After collection, the samples were stored in a freezer (−20 °C) until analysis.Before the analysis, the adsorbent materials were transferred to glass test tubes. The upper section—glass fiber filter, Tenax resin, and polyurethane foam (PUF)—were moved to a test tube and the lower section—Tenax resin and PUF—to another tube. Methanol 2 ml with an internal standard  was added, and the mixture was extracted in an ultrasonic bath for 15 minutes. After extraction, the mixture was filtered through a 0.45 µm filter  into a glass vial.The standards were made by spiking a known quantity of the standard solution into the OVS-sampler and drawing air through the sampler for 5 minutes at a flow rate of 100 ml/min. Standards were extracted and treated as samples.The analysis was performed using a liquid chromatography tandem-mass spectrometry (LC-MS/MS) technique. The instrumentation used was Thermo Finnigan Surveyor liquid chromatography with Thermo Quantum Ultra triple quadrupole mass spectrometry. The analytical conditions were as follows: 100 mm × 2.1 mm, 3.5 µm Waters XTerra C18 column, injection volume of 10 µL, and the mobile phase with a flow rate of 0.3 ml/min. The mobile phase consisted of aqueous 5% methanol with 0.1% formic acid (eluent A) and aqueous 95% methanol with 0.1% formic acid (eluent B), gradient program: 0–30 min, 50% B; 30–42 min, from 50% to 100% B; 42–43 min from 100% to 50% B; 43–50 min 50% B.The standards and samples were scanned through an m/z area of 100–600. All the monitored phthalates occurred as sodium adducts. The ions selected quantification were m/z 441 for DiNP, m/z 469 for DiDP and DPHP and m/z 445 for D4-DiNP.ss corresponds the urinary level of the substance at steady state, D is the external dose as mg/kg bw , BW is the body weight (70 kg), V24 is the estimated average 24-hour urinary volume (1.7 L), and Fue is the mass fraction of the metabolite excreted into the urine during 48 hours. For cx-MiOP, a Fue of 8% was used, based on the study by Anderson et al. [ue of 8% was also used for OH-MPHP, based on the study by Leng et al. [We compared the measured biomarker levels to the biomonitoring equivalents (BEs) calculated on the basis of published health-based limit values, as described by Angerer and co-workers . The basg et al. We studied the occupational phthalate exposure of plastics workers in four different factories. The biomonitoring results of the workers were normalized to a specific gravity in order to take the urinary dilution into account. The profiles of the urinary phthalate metabolite concentration of the workers were also similar when creatinine-adjusted concentrations were used (data not shown).It should be noted that in the present work, it was not possible to differentiate exposure to DPHP and other phthalates containing ester chains of 10 carbons (like DiDP). Accordingly, the biomonitoring results of monohydroxy propylheptyl phthalate  represent the urinary concentrations of all the hydroxy metabolites of the C10-phthalates.The cable factory produced polyvinyl chloride (PVC)-coated cables that contained either DPHP or DiDP as a plasticizer. All five workers worked in the same factory warehouse operating, for example, two extruders. The workers were near the extruders, almost only when the process was about to start and end. The temperature of the closed process was 160–180 °C. Emissions in the air occurred almost entirely only at the points of the extruder line where the cable partly crossed the open cooling line, and local exhaust ventilations (LEVs) were located at these points. The workers wore protective clothing and occasionally also protective gloves (when handling hot machine parts). Respiratory protective equipment (RPE) was not used.n = 60) are provided for comparison [On the sampling day, the DPHP was used as a plasticizer. mparison . The uriNo occupational DPHP exposure data have been published earlier . Gries, As mentioned above, DiDP was also occasionally used in the cable factory. Thus, whether the urinary monocarboxy isononyl phthalate (cx-MiNP) concentration in the urine of workers differed from that in the urine of the control population was of interest. Ninety-two percent of the urinary cx-MiNP concentrations of non-exposed controls were below LOQ—the maximum concentration was 3.4 µg/L . A detec3 (DPHP) and 0.07 mg/m3 (DiDP). The respective results of the second extruder were 0.15 mg/m3 and 0.02 mg/m3. The results showed some emission of DPHP in the second extruder, regardless of the LEV. After conversion, the 8 h time-weighted average (TWA) value was 0.03 mg/m3. This was only 2% of our estimated DNELworkers of 1.45 mg/m3 . Both DPHP and DiDP were analyzed. The results of the first extruder were <0.01 mg/mg/m3 see  and two effects, ). The dieffects, , whereasIn this company, PVC plastics are manufactured using both DPHP and DiNP as plasticizers (in different products though). Four of the five workers mixed PVC resins and additives, and granulated and packed the products. One worker worked on product development in the laboratory and performed small scale mixing. The production procedures were almost entirely automatized and closed, and the workers were near the machines only when starting and ending the process and when cleaning the machines. The workers used protective clothing and gloves—however, some of them wore short-sleeved shirts. The laboratory worker occasionally used nitrile gloves. RPE was not used.All the urinary monoisononyl phthalate (MiNP) results were below LOQ, but monocarboxy isooctyl phthalate (cx-MiOP) concentrations were detected in almost all the urine samples . This waIt should be noted that the DiNP used at the company also contained small amounts of C10-phthalates (like DiDP and DPHP). Although we assume that the OH-MPHP results  are mainOccupational exposure to DiNP also occurred in a German car factory . The med3. This indicated that the small-scale occupational exposure, especially in the case of DPHP, was mainly from skin contact (including hand-to-mouth exposure).Three air samples were collected at the plastics producing company. In this case too, the workers were only occasionally present at the production lines, thus we decided to collect only static air samples. Two samples were collected in the factory and one in the laboratory. One of the factory samples was collected by the granulating machine and the other at the site at which the raw materials feeding took place. The DPHP and DiNP concentrations in all the three air samples were below LOQ 0.01 mg/mThis company manufactured PVC-coated textiles and used DiNP and DPHP as plasticizers. Only DiNP was used during the sampling date. Eight workers took part in the study—six of them working in the factory, one in the laboratory, and one was a production manager. The factory workers prepared the coating material (two workers), operated an extruder (two workers), and other machines (two workers). Protective clothing but no RPE was used.The primary metabolite of DiNP (MiNP) was not detected in any of the workers’ urine samples, which was also the case with the control population samples. The secondary metabolite of DiNP (cx-MiOP), on the other hand, was detectable in almost all the urine—only in two post-shift samples was cx-MiOP concentration below the LOQ . The medThe highest cx-MiOP concentrations were detected in the samples of one extruder operator. The post-shift evening and next-morning samples of one machine operator also contained cx-MiOP concentrations slightly above the P95 level of the control population.As mentioned above, DPHP was also occasionally used in the factory. The after-holiday samples of two workers contained a small amount of OH-MPHP, but their other samples did not contain this metabolite. A small amount of OH-MPHP was detected in the urine samples of one extruder operator (not detected in the after-holiday sample), and in the post-shift evening and next-morning samples of one machine operator. The maximum OH-MPHP concentration of all the samples was 5.2 µg/L. No detectable amounts of OH-MPHP were found in the urine samples of the control population . Accordi3. Once again, this indicated that the small-scale occupational DiNP exposure was mainly from skin contact.DiNP and DPHP concentrations were also analyzed in the air samples collected at the factory. PBZ samples were collected from five of the six factory workers, and one static sample was collected near the control room of the extruder. All the air concentrations were below LOQ 0.01 mg/mThis company reprocessed PVC-coated tarpaulins—the tarpaulin material was manufactured elsewhere. The PVC plasticizer was DiNP. Two workers processed tarpaulins  in a hangar space. Short-sleeved working clothes were used without any PPE.All the MiNP results were below the LOQ. All the urine samples of the workers contained detectable amounts of cx-MiOP (range 6.9 to 33.2 µg/L). The highest concentration was in one after-holiday sample. The level of DiNP exposure was the same as that of the non-occupationally exposed control population.3 of DiNP .One stationary air sample collected at the company contained <0.02 mg/m3 for inhalation exposure. We have previously calculated a biomonitoring equivalent (BE) of 250 µg/L for cx-MiOP corresponding to daily exposure of 0.075 mg/kg [ue of 8% was used as an estimate of the proportion of the cx-MiOP excretion in the urine. No authorities have given any health-based limit values for DPHP, but Bhat et al. [ue of 8% was used as an estimate of the proportion of the OH-MPHP excretion in the urine. Neither ECHA nor Bhat et al. [3 and 1.45 mg/m3 and BEs 900 and 700 µg/L for DiNP (cx-MiOP) and DPHP (OH-MPHP), respectively.ECHA evaluated the scientific data on the toxicity of DiNP in 2013 . Althoug75 mg/kg . This wat et al.  have prot et al. . A Fue ot et al.  derived t et al. . The resWhen these were compared to the measured urinary phthalate levels, we could conclude that even the maximum levels remained well below any of the BE values see . This suThis study presents data on the internal exposure to DiNP and DPHP in four companies that use phthalates in their PVC production or reprocessed PVC plastics. In total, 20 workers took part in the research, and five urine samples were collected from each of them: After-holiday, pre-shift, post-shift, evening, and next-morning samples.In both the cable factory and the plastic producing company, the urinary OH-MPHP concentrations of the after-holiday and pre-shift samples were mostly below the LOQ at 1.0 µg/L. Most of the post-shift, evening, and next-morning urinary concentrations of the workers were above the LOQ (maximum 21 µg/L). Given that all the urinary OH-MPHP concentrations of the non-exposed control population were below the LOQ , these rDiNP was also used in the plastic producing company. Urinary cx-MiOP concentrations were detected in almost all the urine samples of the workers, which was in line with the control population data (98% of the urinary cx-MiOP data were above the LOQ [Two workers who processed tarpaulins were not occupationally exposed to DiNP. The air sample collected at the company contained a DiNP concentration below the LOQ.As is typical with occupational exposure studies, the number of participants in this study was relatively low. However, the results increase our current knowledge on the exposure to these phthalates in plastic product manufacturing and suggest that under these operating conditions, exposures remain low; well below the biomonitoring equivalents estimated on the basis of available health-based limit values. However, further data are needed to obtain a broader overview of occupational exposure to these phthalates in different exposure scenarios.


Second-generation AR antagonists are effective against some of these castration-resistant prostate cancers, but their structural variety is still limited. In this study, we designed and synthesized 4-methyl-7-coumarins as AR antagonist candidates and evaluated their growth-inhibitory activity toward androgen-dependent SC-3 cells. Coumarinamides with a secondary amide bond did not show inhibitory activity, but their N-methylated derivatives exhibited AR-antagonistic activity. Especially, 19b and 31b were more potent than the lead compound 7b, which was comparable to hydroxyflutamide (2b). Conformational analysis showed that the inactive coumarinamides with a secondary amide bond have an extended structure with a trans-amide bond, while the active N-methylated coumarinamides have a folded structure with a cis-amide bond, in which the two aromatic rings are placed face-to-face. Docking study suggested that this folded structure is important for binding to AR. Selected coumarinamide derivatives showed AR-antagonistic activity toward LNCaP cells with T877A AR, and they had weak progesterone receptor (PR)-antagonistic activity. The folded coumarinamide structure appears to be a unique pharmacophore, different from those of conventional AR antagonists.First-generation nonsteroidal androgen receptor (AR) antagonists, such as flutamide ( We have previously reported on the unique conformational properties of aromatic amide compounds . Al. Al30]. 1H and 13C NMR spectra were recorded on a JEOL ECA 600 , or Bruker 600 spectrometer . Mass spectral data were obtained on a Bruker Daltonics microTOF-2focus , Thermo Scientific Q-Exactive or Waters Q-TOF Premier in the positive ion detection mode .All reagents were purchased from Sigma-Aldrich Chemical Co. , Tokyo Kasei Kogyo Co. , Wako Pure Chemical Industries , or Kanto Kagaku Co., Inc. . Silica gel for column chromatography was purchased from Kanto Kagaku Co., Inc . ο-Anisoyl chloride  was added to a solution of 7-amino-4-methylcoumarin  in pyridine (3 mL), and the mixture was stirred at 50 °C for 2 h. The mixture was poured into 2 M HCl (30 mL), and the precipitates were collected, washed with water, and a mixture of chloroform and methanol to give 23a .n-hexane. A solution of 23a  in DMF (1 mL) was added to a suspension of sodium hydride in DMF (2 mL). After 20 min, iodomethane  was added to the mixture. After 5 h, the reaction mixture was poured into water and extracted with ethyl acetate. The organic layer was washed with brine, dried over magnesium sulfate, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate:hexane = 1:1) to give 23b .Sodium hydride  was washed with 2. Then, 10 μL of medium was removed from each well and replaced with 10 μL of drug solution supplemented with serial dilutions of test compound or DMSO as a dilution control in the presence of 1 nM DHT, and the plates were incubated for 3 days. The cell number was determined using a Cell Counting Kit-8  according to the manufacturer’s instruction. This parameter is proportional to the number of living cells in the culture. IC50  according to the manufacturer’s instruction. This parameter is proportional to the number of living cells in the culture. All experiments were performed in triplicate or more.LNCaP cell growth inhibition assay was performed as the previous report ,28,31. B2 for 24 h. Then, 10 μL of medium was removed from each well and replaced with 10 μL of drug solution supplemented with serial dilutions of test compound or DMSO as a dilution control in the presence or absence of 1 nM progesterone, and incubation was continued for 24 h. Then, the medium was aspirated, and the cells were fixed with 100 μL of 1.8% formalin in PBS. The fixed cells were washed with PBS, and 75 μL of assay buffer  was added. The mixture was incubated at room temperature with shielding from light for 2 h, and then the reaction was terminated by the addition of 100 μL of NaOH. The absorbance at 405 nm was measured with a microplate reader.Alkaline phosphatase assay was performed based on the previous report ,32. BrieN-alkyl-arylcarboxamido)coumarins as AR antagonist candidates, using compound 7b as a lead compound. The AR-antagonistic activity of the synthesized coumarinamide derivatives was examined in terms of growth-inhibitory activity toward SC-3 cells. Coumarinamides with a secondary amide bond were inactive, but their N-alkylated derivatives exhibited AR-antagonistic activity. Among them, compounds 19b and 31b showed more potent AR-antagonistic activity than the lead compound 7b, and they were comparable in potency to hydroxyflutamide (2b). Conformational analysis and docking study revealed that the folded structure of coumarinamides associated with the cis-amide bond is important for binding to AR. The selected coumarinamide derivatives showed AR-antagonistic activity toward LNCaP cells with T877A AR and had weak PR-antagonistic activity. The detailed action mechanism of novel coumarinamides as AR modulators including AR-binding ability and transcriptional regulation should be elucidated in the future works, as the folded coumarinamide is a different pharmacophore from those of conventional AR antagonists, and these compounds may be promising candidates for the treatment of prostate cancer.We synthesized a series of 4-methyl-7-(


Atrial remodeling with fibrosis has been well-described in patients with atrial fibrillation (AF). We hypothesized that the left atrial (LA)-late gadolinium enhancement (LGE) extent on cardiac magnetic resonance (CMR) imaging is associated with LA pressure and can be a marker for suitable candidates for non-paroxysmal AF ablation. A total of 173 AF patients with an LA-LGE area on CMR imaging were enrolled. The clinical parameters, including invasively measured LA pressure, were compared between the patients with extensive LA-LGE  and those with small LA-LGE . The E-LGE group had higher peak LA pressures than the S-LGE group . The E-LGE group had more patients with non-paroxysmal AF (non-PAF) (51% vs. 34%), heart failure (9% vs. 0%), and higher NT pro-B-type natriuretic peptide (472 vs. 265 pg/ml) . LA pressure ≥ 21 mmHg was an independent predictor of E-LGE . In the paroxysmal AF (PAF) subgroup, freedom from atrial arrhythmia after catheter ablation was not different . However, in the non-PAF subgroup, it was significantly higher in the S-LGE group than in the E-LGE group . Increased LA pressure was related to the LA-LGE extent. LA-LGE was a good predictor of outcome after catheter ablation, but only in patients with non-PAF. The clinical consequences of AF are increased risks of cardiac morbidity and mortality driven by stroke and congestive heart failure (HF), and it is associated with decreased quality of life3. The catheter ablation of AF is an important treatment modality for patients with drug-refractory AF. However, the reported success rates of AF ablation vary widely, ranging from 40 to 80%4. Despite recent developments in mapping and ablation tools, ablation fails to maintain long-term, sinus rhythm in a substantial number of patients. The guidelines for AF treatment remain controversial, and better patient selection criteria for ablation are needed. Atrial fibrosis is a determinant in the success of rhythm control strategies, including catheter ablation6, and is a marker of atrial remodeling and considered a cause of AF perpetuation. Identification of left atrial (LA) fibrosis is crucial to deciding on the optimal treatment strategy for AF. The role of cardiac magnetic resonance (CMR) has received attention for selecting the best candidates for ablation7.Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia, with prevalence increasing along with the age of the populationLate gadolinium enhancement (LGE) CMR is a non-invasive imaging technique that assesses the structure and tissue composition of the heart. However, the clinical role of CMR imaging in patients with AF has not yet been fully established. The atrial wall myocardium is thin, and irregular ventricular rhythms complicate functional analysis during AF. Furthermore, the implications and formative processes of LA-LGE remain to be elucidated.We sought to evaluate the association between LA pressure and the LA-LGE extent in CMR images in AF ablation candidates. The clinical value of LA-LGE on CMR imaging as a predictor of atrial arrhythmia recurrence was investigated in patients who underwent catheter ablation.We prospectively enrolled consecutive patients referred for catheter ablation for drug-refractory AF. Only patients without a previous ablation for AF and without contraindications for CMR were included. LGE-CMR, transthoracic echocardiography (TTE), and transesophageal echocardiography (TEE) were performed within 24 h prior to catheter ablation. All participants provided written informed consent. The clinical type of AF was categorized as paroxysmal AF  or non-PAF (AF lasting more than 7 days or needing cardioversion for termination).The study population was divided into two groups based on the percentage of LA-LGE. The LGE area was calculated as described below. The extensive LGE (E-LGE) group included patients with an LGE extent ≥ 20%, of the mean LA-LGE value of all patients and the small LGE (S-LGE) group included those with LGE extents < 20%. We compared the clinical and functional parameters between the two groups to investigate the pathogenesis of LA-LGE. The study design flow chart and a representative case from both groups are shown in Fig. This study was performed in accordance with the ethical standards of the 1964 Declaration of Helsinki. The study protocol was approved by the Institutional Review Board of Korea University Medical Center.9. CMR was performed using a 3-T MR system  with a 32-element phased-array cardiac coil. MR angiography of the pulmonary veins (PVs) was obtained using a contrast-enhanced timing robust angiography sequence after an injection of 0.2 mmol/kg gadolinium-based contrast . High spatial resolution LGE-CMR images were acquired approximately 20 min after the injection of the contrast agent using a 3-dimensional (3D) inversion recovery-prepared, respiration-navigated, electrocardiography (ECG)-gated, gradient-echo pulse sequence after the Look-Locker sequence to identify the optimal nulling time for the normal left ventricular myocardium. The typical acquisition parameters of the LGE-CMR images were as follows: voxel size, 1.5 mm × 1.5 mm × 1.5 mm; repetition time/echo time, 4.7/1.4 ms; inversion time, 230–270 ms; flip angle, 25°; bandwidth, 127 Hz/pixel; one RR interval between the inversion pulses; phase encoding direction in the right-left; and a parallel imaging technique using sensitivity encoding with R = 2. All LGE-CMR images were transferred into the workstation for quantitative analysis.The LA-LGE CMR protocol and the LGE quantification method were described in our previously reported studyThe LGE-CMR images were analyzed using Terarecon iNtuition software  by a radiologist (SHH) who was blinded to the LA pressure and other clinical information. On each coronal or transverse LGE-CMR image, the endocardial and epicardial borders of the LA wall were semi-automatically contoured to select the LA wall. A 3D LA model was reconstructed by combining the entire LA wall voxels. In the LA wall, the voxels of enhancement were identified using the full width at half-maximum (FWHM) technique with a manual delineation of the regions of interest (ROI) focusing on the mitral valve leaflet. The ROI was drawn around the mitral valve leaflet with high signal intensity to define the maximum signal for the FWHM technique, which provided the signal threshold as one-half of the maximum signal from the mitral valve leaflet. The LA wall pixels with signal intensities greater than the signal threshold of LGE were automatically selected, and defined as the LA-LGE pixels. After the identification of the LA-LGE pixels over the entire slices, a color look-up table mask was applied yellow to the LA-LGE pixels and the white to other pixels on the 3D LA model for better delineation of the LA wall composition. To assist in the process, the initial visualization used a volume-rendering tool in CORVIEW (Marrek Inc) that allowed the distribution of enhancement in 3D. Then, the LA wall voxels of enhancement were defined as LA-LGE. The sum of the LA-LGE over all of the LGE-CMR images was considered the LA-LGE volume (in ml). Furthermore, the extent of LA-LGE was calculated as the percentage of the LA-LGE volume in the total LA wall volume Fig. .9. Briefly, a radiologist assessed the LA areas on the perpendicular planes of the 2- and 4-chamber CMR images. The LA axis length from the mitral annulus level to the posterior wall of the LA was evaluated in the 2-chamber cine CMR images. The biplane area-length technique for the calculation of LA volume was based on this formula: LA volume (ml) = (0.85 × LA area on the 4-chamber cine CMR image × LA area on the 2-chamber CMR image)/LA axis length in the 2-chamber cine CMR image. We calculated the LA volumes based on the LA areas and LA axis length was determined from the cine CMR images. The LA volume measurements were indexed to the body surface area. The left ventricular (LV) ejection fraction (EF), E/Em ratio, and right ventricular systolic pressure were measured by TTE. The presence of spontaneous echo contrast and thrombus was evaluated by TEE.ECG-gated cine CMR was performed to evaluate the LA function. The cine CMR images were analyzed according to a previously published methodBlood samples were collected from the peripheral vein of each patient within 24 h prior to the LGE-CMR. NT pro-B-type natriuretic peptide (BNP), von Willebrand factor (vWF), D-dimer, and high-sensitivity C-reactive protein (hs-CRP) were measured to evaluate their relationships with the LA-LGE extent. We compared the biomarkers between the E-LGE and S-LGE groups.All patients fasted 8 h prior to catheter ablation. Saline fluid (0.9%) was injected during that time at a rate of 40 ml/h. Sedation was used during the procedure and pressure measurements. The LA was assessed via transseptal puncture using a long sheath  Fig. A. The LAThe circumferential antral ablation of 4-PVs was performed guided by a NavX 3-dimensional mapping system . The endpoint of PV ablation was eliminating all PV potentials. The radiofrequency energy was delivered with a power of 30 W and a target temperature of 48 ℃  using a 4-mm open irrigated-tip catheter . The subsequent strategy was different according to clinical type of AF. The procedural endpoint was eliminating all triggers, in addition to PV isolation (PVI) in patients with PAF. That endpoint in patients with non-PAF was termination and no further induction of sustained atrial arrhythmia. If AF was not terminated after PVI, LA complex fractionated atrial electrograms (CFAEs) were mapped. If AF was organized to AT during PVI or CFAE ablation, activation mapping-guided ablation was performed. If SR was not restored after the ablation of the aforementioned lesions, direct-current electrical cardioversion was used.Follow-up rhythm examinations and 12-lead ECGs were performed at 1, 3, 6, 9, and 12 months after the procedure. Holter or 30-day event recorder monitoring was performed at 3, 6, and 12 months. Recurrence as a clinical outcome was defined as any episode of AF/AT lasting more than 30 s after a blanking period of 3 months. Antiarrhythmic drugs (AADs) were continued for 1–3 months after the procedure and discontinued at the end of the blanking period.t test was used to compare the continuous variables between the E-LGE and S-LGE groups, and the categorical variables were compared by Chi-squared test or Fisher’s exact test. Multivariate analysis was conducted with a logistic regression model reporting odds ratios (OR). The predictor variables included age, female, AF type, diagnosis to ablation time, body mass index, CHA2DS2 VASc ≥ 2, LA pressure ≥ 21 mmHg, the LA volume index, LV EF, NT pro-BNP, and E/Em ratio. Multiple regression analysis was performed using the criterion of p < 0.10 in univariate analysis for the inclusion of a variable in the model. The time to AF/AT recurrence was measured using Kaplan–Meier analysis with comparisons by log-rank statistics. A p-value of less than 0.05 was considered significant statistically. SPSS 20.0 software  was used for all statistical analyses.Continuous variables are expressed as the mean plus or minus the standard deviation. Categorical variables are reported as counts with percentages. An independent Student’s Between December 2014 and September 2015, 194 consecutive patients with drug-refractory AF were referred for catheter ablation of AF. None had a history of AF procedures or thoracic surgery, and none had significant mitral valve disease. After excluding 13 patients who declined consent or could not undergo CMR, 181 patients underwent TTE, TEE, and LGE-CMR within 24 h prior to ablation. In this study, six patients were excluded because of unacceptable quality of CMR images among the patients underwent CMR. During the LGE image acquisition, the mean heart rate of all of the patients was maintained below 100 beats per minute. One patient with cardiac tamponade before measurement of the LA pressure and another patient who failed AF cardioversion were excluded. Ultimately, 173 patients  were included in the analysis. The LA-LGE area measured by CMR imaging showed a maximum of 58% and a mean of 19%. The E-LGE group contained 78 patients (45%), and the S-LGE group contained 95 patients (55%). A comparison of the baseline characteristics between the groups is provided in Table 2, p = 0.085) tended to be higher in the E-LGE group than in the S-LGE group. The LV EF  and E/Em ratio  measured by TTE were not different between the two groups. The existence of spontaneous echo contrast was not different between the groups.The LA anatomical data and function were analyzed by CMR imaging and echocardiography Table . The meaThe LA pressure measurements were compared between the groups. It was significantly higher in the E-LGE group compared to the S-LGE group   was significantly greater in the E-LGE group  Table . Von WilThere was no recurrence of atrial arrhythmia in 81.1% of the S-LGE group and 69.7% of the E-LGE group after ablation over 1 year of follow-up (p = 0.060). The estimated recurrence-free survival is shown in Fig. This study showed that (1) the LA-LGE extent on CMR imaging was associated with clinical HF and elevated LA pressure and (2) the E-LGE group showed worse outcomes after catheter ablation, especially in patients with non-PAF.10 and clinical outcomes of RFCA for AF11. Atrial fibrosis revealed by LGE-CMR imaging is useful in identifying optimal candidates for procedures to treat AF. These findings suggest that CMR imaging improved the selection process and outcome for patients considered for AF ablation. However, the clinical application of LGE-CMR imaging assessment is still limited because of poor reproducibility in different centers.CMR imaging in patients with AF has the advantage of providing an exact detailed image of the LA and surrounding structures. Furthermore, CMR imaging can assess the reservoir, conduit, and contractile function based on phasic changes in volumes. The LA-LGE in CMR images showed the degree of LA fibrosis. In addition, the LA-LGE extent in CMR imaging can non-invasively predict stroke risk13 and is correlated with the scar area in electrophysiologic studies of patients with AF. In our study, patients with extensive LGE (E-LGE group) had high rates of HF history, increased NT pro-BNP, and high LA pressure. These markers are associated with clinical heart function and atrial remodeling. Atrial fibrosis could either represent the cause or result of physiologic dysfunction. Habibi et al. showed that LA-LGE in feature-tracking CMR imaging was associated with LA function independent of age, sex, hypertension, type of AF, LA size, and wall thickness14. In the present study, the LA volume did not differ between the groups although it is a well-known marker of LA remodeling. Chrispin et al. reported that the pre-ablation LA-LGE extent was associated with increased LA volume. However, the strength of the relationship was weak in ablation candidates15. Early-stage remodeling with atrial fibrosis in LA did not show certain LA dilatation. We showed that atrial fibrosis was associated with the directly measured LA pressure. Increased LA pressure is a marker of diastolic dysfunction of the heart. Prior studies have hypothesized that atrial fibrosis is the result of atrial dilatation and diastolic dysfunction as evaluated by imaging studies, such as echocardiography or CMR.LA-LGE is a marker of atrial fibrosis16. The LA pressure is the output of LV diastolic function, and LV diastolic dysfunction can increase atrial afterload. Peak LA pressure occurs during the early filling phase of the atrium. Atrial stretch and dilatation to compensate for the rising atrial wall stress subsequently result in AF17. The LA can compensate for these demands through the generation of more extracellular matrix, with the expression of proteins such as collagen. With increasing atrial wall stress, atrial fibrosis progressively creates a substrate for AF18. Hemodynamic stress, such as ventricular diastolic dysfunction, causes the development of fibrosis rather than hypertrophy in the atria since the thin-walled LA responds to pressure overload with dilatation rather than wall thickening19. Atrial fibrosis impairs inter-myocyte coupling via gap junctions, resulting in fragmented conduction that promotes arrhythmias20. Some prior reports have suggested a relationship between atrial stretch and AF substrates. LA pressure directly affects the maintenance of AF by mechanoelectrical feedback or stretch-induced pro-fibrotic reactions, creating AF substrates. LA pressure-independent atrial dilatation also increases the critical mass that begets AF.Increased LA pressure provokes the occurrence and maintenance of AF22. The LA-LGE in CMR imaging reflects atrial anatomical and physiologic remodeling and may be a surrogate marker of the clinical AF type for ablation strategy decisions.The LA-LGE in CMR imaging offers a new predictor of clinical outcome after catheter ablation, reflecting LA anatomical remodeling with fibrosis. The current ablation strategy is mainly decided based on the clinical type of AF, i.e., paroxysmal or non-paroxysmal. However, LA remodeling with fibrosis did not closely correspond to the type of AF in this study. The non-PAF patients with low-grade LA-LGE showed good outcomes similar to those of PAF patients (more than 80% with AF/AT freedom rates over 1 year). A well-known LGE-CMR study in AF patients, DECAAF , also showed a weak correlation between AF type and LA-LGEOur study was limited by its single-center, observational design. In addition, routine performance of quality 3D LGE-CMR imaging studies in patients requires a high level of expertise in CMR imaging and support during the studies by expert MR technologists and imagers. Processing of the CMR images is laborious and requires experienced observers to perform LA wall tracings and select the threshold levels. Improvements are expected to simplify and further standardize image processing. The accurate quantitative analysis of LA-LGE also remains a challenge. The walls of the LA are thin (1–2 mm) and susceptible to a partial volume effect from the surrounding compartments, like blood or the esophagus. To attenuate the inter-observer error of the quantification method, the LA-LGE volumes were measured twice by two independent investigators. LA pressure is not a fixed parameter because it is sensitive to the volume and heart rate of the patient. We measured the LA pressure at the beginning of the procedure after overnight fasting for 8 h. To prevent any impact from LA stunning, we measured the LA pressure 5 min after cardioversion. The catheter designed to carefully measure the pressure was not used. Finally, the image quality of LGE image in the quantification of LA wall composition is often confused by arrhythmia and it can impact the result of this study. We initially excluded the patients with unacceptable quality of CMR images. The impact of rhythm status during CMR to result was not considered because LGE amount and procedural outcome were not related to rhythm status during CMR in patients with PAF. In addition, the most non-paroxysmal AF patients underwent CMR during AF status.LA-LGE in CMR imaging was associated with LA remodeling and clinical HF. Increased LA pressure was related to the extent of LGE in the LA. LA-LGE was a good predictor of outcome after catheter ablation, but only in patients with non-PAF.Supplementary Figures.


Given the high similarity of phenotypical and secretory properties of mesenchymal stem cells and fibroblasts, this study investigated the possibility of inducing EMT process by mesenchymal stem cells.Annually, more than 13% of deaths worldwide occur due to cancer. One of the main reasons for the high mortality rate is due to the metastasis of cancer stem cells. Induction of metastasis occurs during the EMT process, which can also be stimulated by fibroblast cells.Mesenchymal stem cells (MSCs) were isolated and sub-cultured until passage 3 or 4. AGS cells were co-cultured with MSCs for 4 days. As the positive control group, AGS cells were treated with TGF-β (10ng/ml) for 48h. Finally, the mRNA expression level of Vimentin, β-catenin, Snail, and E-cadherin as the EMT pattern, were evaluated by RT-PCR technique. Our findings indicated that AGS cells’ crosstalk with MSCs significantly upregulated fibroblast markers including Vimentin and Snail expression. However, no significant changes were identified for β-catenin gene expression. Additionally, AGS treatment with MSCs resulted in diminished E-cadherin in the targeted cells. Based on the results, the AGS cells crosstalk with MSCs activates induction of epithelial mesenchymal transition, which is confirmed through the elevation of Vimentin and Snail expression and reduction of E-cadherin expression as a specific epithelial marker. However, it seems that MSc was not effective on Wnt/ β-catenin signal gastric cancer cell line. Gastric cancer is a multifactorial process recognized as one of the leading causes of cancer deaths worldwide . Gastrichigh and CD24low cells  was obtained from Research Institute of Gastroentrology and Liver Diseases . It was cultured under exponential growth in complete Roswell Park Memorial Institute medium (RPMI)  supplemented with 15% FBS, 1% penicillin /streptomycin, 1% non-essential amino acids , and 1% L-glutamine , and cultured in a 5% humidified CO2 incubator at 37°C.Induction of Epithelial-Mesenchymal Transition by AGS-MSCs co-culture system5 AGS cells in complete RPMI medium supplemented with 10% FBS and 1% penicillin /streptomycin were added to the basal surface of each insert plate. Next, 150000 MSCs in DMEM medium, containing 10% FBS, and 1% penicillin/ streptomycin were transported to the upper surface of each well. Eventually, after 3 days, the AGS cell were detached by Trypsin-EDTA and utilized for subsequent tests.Once the cells reached the appropriate confluency, the MSCs and AGS cells were trypsinized , then counted for transformation to a co-culture insert plate 0.4 µm . For this purpose, approximately 2×10Treatment of AGS by TGF-β for inducing Epithelial-Mesenchymal Transition5 per well, in 6-well plates in an RPMI medium supplemented with 10% FBS and 1% penicillin /streptomycin and incubated at 37 °C with 5% CO2 for 24 hours. Subsequently, to induce cell differentiation, cell growth decreased by reducing FBS to 0.5% as serum starvation. Simultaneously, 10 ng/ml of TGF-β was added to the culture media. After 48 hours of treating with TGF-β (10ng/ml), the cells were collected and used for the following tests.AGS cells treated with TGF-β1 were used as the control group to be compared with the AGS-MSCs co-culture system. AGS cells were seeded almost 2×10RNA extraction and cDNA synthesisTotal RNA was isolated by RNA Purification Mini Kit , based on the manufacturer's instructions. The concentration of the total RNA was determined via the Nanodrop  by absorbance at 260 nm. Subsequently, the extracted RNA was converted to cDNA, according to RevertAid First Strand cDNA Synthesis Kit  using a random hexamers primer in the presence of RNase inhibitor. The quality and concentration of cDNA were measured by Nanodrop Device. The synthesized cDNA was stored at -70 °C until further analysis.Quantitative real-time PCRTo perform quantitative real-time PCR reaction, initially, the primers of the desired genes for assessment of EMT induction  and internal control gene (GAPDH) were designed by primer 3 software and blasted at NCBI. For validation of the accuracy and specificity of the primers, Gene Runner (ver. 6.0.04) was used. The sequence of the primers is shown in Statistical analysisPrism software statistical package version 5  was utilized for data analysis. Independent t-test was applied for examining the differences between the experimental groups with the data presented as mean (SEM). Significance was set as the difference P-value is less than 0.05Phenotypical assessmentAGS-MSCs crosstalk affects the specific makers of EMT.Once treated with MSCs for 3 days, the morphological changes of AGS cells were observed with an inverted microscope . The cells have a plating efficiency of approximately 80% in the medium at passage number 3. The AGS- MSCs treated cells had remarkable phenotypic differences from the AGS cells without any treatment as the control group . CompareTo investigate the impact of MSCs-secretions on induction of EMT, qRT-PCR was performed to detect the expression of Vimentin, β-catenin, Snail, and E-cadherin on targeted cells. The AGS cells without any treatment were used as negative control group while the AGS cells treated with TGF-β were utilized as positive control group. AGS-MSCs crosstalk considerably elevated the expression of EMT markers gene, including Vimentin, β-catenin, and Snail. The expressions of Vimentin and Snail genes were significantly upregulated when AGS cells were co-cultured with MSCs and TGF-β treated groups in comparison with the control group without any treatment . Based oMSCs are one of the main subsets of adult stem cells, which can be separated from varied tissues such as adipose tissue, bone marrow, and cord blood. Numerous studies have reported that MSCs have unique characteristics such as high proliferation and differentiation capacity, ease of access and separation, and potential of secretion of various components including growth factors and cytokines . Hence, Additionally, E-cadherin expression level was reduced significantly in the AGS cells co-cultured with MSCs. Epithelial cells are detectable by E-cadherin expression where the disappearance of this marker is a major sign of EMT occurrence . Taken tInconsistent with our study, a previous study by Hidehiko Takigawa et al. reported that the potential of the proliferation and migration of KM12SM colon cancer cells increased in direct co-culture with MSCs. They observed elevation in the expression of EMT-related genes including, fibronectin, SPARC, and galectin 1 following KM12SM- MSCs co-culture via microarray analysis. Furthermore, they performed qRT-PCR of EMT-related genes, which provided supportive evidence for microarray results. Hence, their results suggested that MSCs may have a great ability in inducing EMT in colon cancer cells causing colon cancer metastasis .Another study by Sendurai A et al. revealed that MSCs secreted cytokines affected breast cancer cells by altering the expression of genes, including E-cadherin, Vimentin, Twist, and Snail. As mentioned above, these genes are involved in the modulation of epithelial state transition to mesenchymal state and thus developing breast cancer cells capable of migration and invasion (30).In contrast with previous studies, Clarke et al. demonstrated that MSCs decreased the migration and invasion of human breast cancer cells via modulating TIMP-1 and -2 secretion .Overall, in the current study, the induction of EMT was examined by qRT-PCR in three experimental groups . Our results revealed that co-culturing AGS cells with MSCs results in overexpression of Snail and Vimentin as well as TGF-β treated group compared to AGS cells alone. Additionally, E-cadherin expression was significantly down-regulated. However, β-catenin expression, in AGS-MSC co-cultured group was not significantly altered in comparison with AGS cells without any treatment. Taken together, MSCs showed a high capacity to induce EMT in gastric cancer cell line.The authors declare that they have no conflict of interest.